Although there is a great deal of information available on the factors which influence the breakdown and resorption of bone, information on growth factors which stimulate the formation of new bone is more limited. Investigators have searched for sources of such activities and have found that bone tissue itself is a storehouse for factors which have the capacity for stimulating bone cells. Thus, extracts of bovine tissue obtained from slaughterhouses contain not only structural proteins which are responsible for maintaining the structural integrity of bone, but also biologically active bone growth factors which can stimulate bone cells to proliferate. Among these latter factors are transforming growth factor .beta., the heparin-binding growth factors (acidic and basic fibroblast growth factor), the insulin-like growth factors (insulin-like growth factor I and insulin-like growth factor II) and a recently described family of proteins called bone morphogenetic proteins (BMPs). All of these growth factors have effects on other types of cells as well as on bone cells.
The BMPs are novel factors in the extended transforming growth factor .beta. family. They were first identified in extracts of demineralized bone (Urist 1965, Wozney et al., 1988). Recombinant BMP-2 and BMP-4 can induce new bone formation when they are injected locally into the subcutaneous tissues of rats (Wozney 1992, Wozney & Rosen 1993). These factors are expressed by normal osteoblasts as they differentiate, and have been shown to stimulate osteoblast differentiation and bone nodule formation in vitro as well as bone formation in vivo (Harris et al., 1994). This latter property suggests potential usefulness as therapeutic agents in diseases which result in bone loss.
The cells which are responsible for forming bone are osteoblasts. As osteoblasts differentiate from precursors to mature bone-forming cells, they express and secrete a number of the structural proteins of the bone matrix including Type-1 collagen, osteocalcin, osteopontin and alkaline phosphatase (Stein et al, 1990, Harris et al, 1994). They also synthesize a number of growth regulatory peptides which are stored in the bone matrix and are presumably responsible for normal bone formation. These growth regulatory peptides include the BMPs (Harris et al, 1994). In studies of primary cultures of fetal rat calvarial osteoblasts, BMPs 1, 2, 3, 4, and 6 are expressed by cultured cells prior to the formation of mineralized bone nodules (Harris et al, 1994). Expression of the BMPs coincides with expression of alkaline phosphatase, osteocalcin and osteopontin.
Although the BMPs have powerful effects to stimulate bone formation in vitro and in vivo, there are disadvantages to their use as therapeutic agents to enhance bone healing. Receptors for the bone morphogenetic proteins have been identified in many tissues, and the BMPs themselves are expressed in a large variety of tissues in specific temporal and spatial patterns. This suggests that they may have effects on many tissues other than bone, potentially limiting their usefulness as therapeutic agents when administered systemically. Moreover, since they are peptides, they would have to be administered by injection. These disadvantages are severe limitations to the development of BMPs as therapeutic agents.
It is an object of the present invention to overcome the limitations inherent in known osteogenic agents by providing a method to identify potential drugs which would stimulate production of BMPs locally in bone.